Pyridine-Containing Octadentate Ligand NE3TA-PY for Formation of Neutral Complex with ¹⁷⁷Lu(III) and ⁹⁰Y(III) for Radiopharmaceutical Applications: Synthesis, DFT Calculation, Radiolabeling, and In Vitro Complex Stability

Abstract

Targeted radionuclide therapy is a developing therapeutic modality for cancer and employs a cytotoxic radionuclide bound to a chelating agent and a bioactive molecule with high binding affinity for a specific biomarker in tumors. An optimal chelator is one of the critical components to control therapeutic efficacy and toxicity of targeted radionuclide therapy. We designed a new octadentate ligand NE3TA-PY (7-[2-[(carboxymethyl)(2-pyridylmethyl)amino]ethyl]-1,4,7-triazacyclononane-1,4-diacetic acid) for β-particle-emitting ¹⁷⁷Lu and ⁹⁰Y with targeted radionuclide therapy applications. The pyridine-containing polyaminocarboxylate ligand was proposed to form a neutral complex with Lu(III) and Y(III). The new chelator NE3TA-PY was synthesized and experimentally and theorectically studied for complexation with ¹⁷⁷Lu(III) and ⁹⁰Y(III). DFT-optimized structures of Y(III)-NE3TA-PY and Lu(III)-NE3TA-PY complexes were predicted. NE3TA-PY displayed excellent radiolabeling efficiency with both ¹⁷⁷Lu and ⁹⁰Y. The new chelator (NE3TA-PY) bound to ¹⁷⁷Lu was more stable in human serum and better tolerated when challenged by EDTA than ⁹⁰Y-labeled NE3TA-PY. Our findings suggest that the new chelator (NE3TA-PY) produced excellent Lu-177 radiolabeling and in vitro complex stability profiles.

Graphical Abstract

Introduction

Molecularly targeted radionuclide therapy using an alpha- or beta-particle emitter has been developed as a therapeutic modality for cancer.¹ Numerous targeted radiotherapeutics built on various radionuclide-biomolecule combinations have been evaluated for treatment of cancer in preclinical and clinical settings.^1–9 Successful radionuclide therapy requires a well-coordinated therapeutic platform consisting of a potent therapeutic radionuclide, a tumor-targeting biomolecule, and a bifunctional chelator for labeling of the biomolecule with radionuclide.^{1,3,4 90}Y (t_1/2 = 64 h) and ¹⁷⁷Lu (t_1/2 = 6.7 d) are potent radionuclides suitable for targeted cancer therapy. Y-90 (t_1/2 = 2.7 d, E_max = 2.3 MeV) is a highly energetic and pure ®⁻-emitting radionuclide with the advantage of a longer range of penetration and homogeneous dose distribution at optimal therapeutic range.^{1,2,7 177}Lu is a β⁻-particle emitter with relatively long half-life (t_1/2 = 6.7 d, E_max = 0.5 MeV) and less energetic and shorter tissue-penetration range (1.6 mm) relative to ⁹⁰Y (11.3 mm).^5,8,9 Therapeutic efficacy of the β-particle emitting radionuclides ¹⁷⁷Lu and ⁹⁰Y has been highlighted in FDA-approvals of two clinically available radiopharmaceuticals, Zevalin® (⁹⁰Y-labeled anti-CD20 antibody) for non-Hodgkin’s lymphoma (NHL) therapy and Lutathera® (¹⁷⁷Lu-labeled tyrosin3-octreotate peptide) for treatment of neuroendocrine tumors (NETs).^1,7–9 Zevalin® is structured on ⁹⁰Y-1B4M-DTPA (2-(4-isothiocyanatobenzyl)-6-methyl-diethylenetriamine pentaacetic acid), while Lutathera® contains enhance therapeutic potency. Various chelators including DOTA and DTPA (diethylenetriamine pentaacetic acid) analogues (Figure 1) have been employed for radiolabeling of tumor-targeting peptides and antibodies with ⁹⁰Y or ¹⁷⁷Lu.^1–3,7–9 In general, the macrocyclic DOTA can form a stable complex with a metal, while the acyclic DTPA rapidly bound to a metal.

Figure 1.

Structure of Chelating Agents DOTA, DTPA, NETA, NE3TA, and NE3TA-PY

We have synthesized and evaluated structurally novel chelating agents in the series of NETA ((7-[2-[bis(carboxymethyl)amino]ethyl]-1,4,7-triazacyclononane-1,4-diacetic acid) and NE3TA (7-[2-(carboxymethyl)amino]ethyl]-1,4,7-triazacyclononane-1,4-diacetic acid) containing both macrocyclic and acyclic binding moieties for rapid formation of a stable complex with therapeutic or diagnostic radionuclides including ⁹⁰Y, ¹⁷⁷Lu, ⁶⁴Cu, and ^205/6Bi.^10–15 We were interested in development of chelation chemistry for formation of a neutral complex with Lu(III) and Y(III). Neutral metal complexes were reported to be more inert to acid/cation-promoted dissociation when compared with ionic complexes.^16–17

We herein report synthesis and evaluation of a NE3TA analogue containing a pyridine (PY) ring (Figure 1) for ¹⁷⁷Lu(III) and ⁹⁰Y(III). The new chelator NE3TA-PY has different acyclic donor groups structured on 1,4,7-triazacyclononane (TACN) and can form 8-coordinate complexes with metal cations using four tertiary nitrogen atoms, two acetate groups, and a nitrogen donor in the pyridine ring. The new chelator is hypothesized to rapidly form a stable neutral complex with the β-particle emitting radionuclides ¹⁷⁷Lu(III) or ⁹⁰Y(III) with a relatively large ionic radius. The new chelator NE3TA-PY was synthesized and evaluated for radiolabeling kinetics and in vitro complex stability with ⁹⁰Y and ¹⁷⁷Lu. The structures of the neutral metal complexes Lu(III)-NE3TA-PY and Y(III)-NE3TA-PY were predicted using DFT methods.

Experimental

Instruments and Methods.

¹H, ¹³C, and DEPT (distortionless enhancement by polarization transfer) NMR spectra of the new compounds were obtained using a Bruker 300 NMR instrument, and chemical shifts are reported in ppm on the d scale relative to TMS (tetramethylsilane). Electrospray ionization (ESI) high resolution mass spectra (HRMS) of the new compounds were obtained on JEOL double sector JMS-AX505HA mass spectrometer (University of Notre Dame, IN). Analytical HPLC was performed on an Agilent 1200 equipped with diode array detector (λ = 254 and 280 nm), with the thermostat set at 35°C and with a Zorbax Eclipse XDB-C18 column (4.6×150mm, 80 Å). A combination of a binary gradient (0–100% B/15min; solvent A = 0.1% trifluoroacetic acid (TFA) in H₂O; solvent B = 0.1% TFA in CH₃CN) at a flow rate of 1 mL/min was used for analytical HPLC.

Reagents.

All reagents were purchased from Sigma-Aldrich (St. Louis, MO) and used as received unless otherwise noted. ⁹⁰Y (0.05 M HCl) and ¹⁷⁷Lu (0.05 M HCl) were purchased from Perkin-Elmer. ⁹⁰Y (t_1/2 = 64.1 h) is a β-emitting radionuclide. ¹⁷⁷Lu (t_1/2 = 6.7 days) is a β/γ-emitting radionuclide. Appropriate shielding and handling protocols should be in place when using the radioisotopes.

4-[2-(Benzyl-tert-butoxycarbonylmethyl-amino)-ethyl]-7-tert-butoxycarbonyl-methyl[1,4,7]triazonan-1-yl)-acetic acid tert-butyl ester (3).

Compound 3 was prepared via a modification of the method as previously reported.¹⁸ To a solution of 1¹⁹ (881.4 mg, 2.46 mmol) in CH₃CN (25 mL) was added diisopropylethylamine (DIPEA, 954.1 mg, 7.40 mmol) and 2¹⁸ (807.4 mg, 2.46 mmol). The reaction mixture was stirred under reflux for 14.5 h. The resulting solution was evaporated in vacuo, and the residue is purified via column chromatography (silica gel, 60 mesh) eluting with 3% methanol in dichloromethane to afford pure 3 as a light yellow oil (1.36 g, 91%). ¹H and ¹³C NMR spectra of compound 3 is identical to those reported in the literature.^{18 1}H NMR (CDCl₃, 300 MHz) δ 1.33 (m, 27H), 2.53–2.79 (m, 4H), 2.81–3.02 (m, 2H), 3.04–3,09 (m, 4H), 3.16 (s,2H), 3.21–3.30 (m, 4H), 3.38–3.49 (m, 2H), 3.52–3.65 (m, 4H), 3.73 (s,2H), 7.26 −7.29 (m, 5H); ¹³C NMR (CDCl₃, 300 MHz) δ 28.1 (q), 49.1 (t), 49.3 (t), 52.2 (t), 52.6 (t), 53.7 (t), 55.3 (t), 57.7 (t), 58.3 (t), 81.5 (s), 81.6 (s), 127.6 (d), 128.5 (d), 129.1 (d), 137.5 (s), 170.3 (s), 170.4 (s).

di-tert-Butyl 2,2’-(7-(2-((3,3-dimethyl-2-oxobutyl)amino)ethyl)-1,4,7-triazonane-1,4-diyl)diacetate (4).

To a solution of 3 (1 g, 1.65 mmol) in EtOH (50 mL) was added 10% wet Pd/C (300 mg). The resulting mixture was subjected to hydrogenolysis at room temperature for 40 h by agitation with excess H₂ (g) at 60 psi in a Parr hydrogenator apparatus. The reaction mixture was filtered through Celite®, and the solvent was evaporated in vacuo to provide compound 4 (830 mg, 97.6%) as a colorless oil. ¹H NMR (CDCl₃, 300 MHz) δ 1.26 (m, 27H), 2.74–3.59 (m, 23H). ¹³C NMR (CDCl₃, 75 MHz) δ 27.9 (q), 44.3 (t), 49.1 (t), 49.9 (t), 50.9 (t), 53.2 (t), 56.9 (t), 57.5 (t), 82.2 (s), 83.2 (s), 167.2 (s), 169.8 (s). HRMS (positive ion ESI) Calcd for C₂₆H₅₀N₄O₆ [M + H]⁺ m/z 515.3803. Found: [M + H]⁺ m/z 515.3798.

di-tert-Butyl 2,2’-(7-(2-((3,3-dimethyl-2-oxobutyl)(pyridin-2-ylmethyl)amino)ethyl)-1,4,7-triazonane-1,4-diyl)diacetate (5).

To a solution of 4 (100 mg, 0.19 mmol) and potassium carbonate (31.5 mg, 0.22 mmol) and sodium iodide (34.2 mg, 0.22 mmol) in anhydrous CH₃CN (2 mL) at 0 °C was dropwise added 2-(chloromethyl)pyridine (24.2 mg, 0.19 mmol). The reaction mixture was filtered and concentrated to dryness to provide compound 5 (103.8 mg, 90.2%). ¹H NMR (CDCl₃, 300 MHz) δ 1.41 (m, 27H), 2.70–2.80 (m, 4H), 3.00–3.75 (m, 18H), 3.93 (s, 2H), 7.23 (t, J = 6.0 Hz, 1H), 7.31 (d, J = 6.0 Hz, 1H), 7.65 (t, J = 9.0 Hz, 1H), 8.51 (d, J = 6.0 Hz, 1H). ¹³C NMR (CDCl₃, 75 MHz) δ 28.2 (q), 49.7 (t), 52.8 (t), 53.0 (t), 54.0 (t), 56.0 (t), 57.9 (t), 59.4 (t), 81.6 (s), 122.6 (d), 123.7 (d), 136.9 (d), 149.3 (d), 157.7 (s), 170.4 (s), 170.7 (s). HRMS (positive ion ESI) Calcd for C₃₂H₅₅N₅O₆ [M + H]⁺ m/z 606.4225. Found: [M + H]⁺ m/z 606.4217.

7-[2-[(carboxymethyl)(2-pyridylmethyl)amino]ethyl]-1,4,7-triazacyclononane-1,4-diacetic acid (6).

To Compound 5 (16.6 mg, 0.027 mmol) was added 6M HCl (3 mL). After addition, the reaction mixture was refluxed for 5 min. The resulting solution was cooled to the room temperature and washed with CH₂Cl₂. The aqueous layer was evaporated in vacuo to afford compound 6 (15.1 mg, 90.2%) as a light yellowish solid. Analytical HPLC (t_R = 8.9 min). ¹H NMR (D₂O, 300 MHz) δ 3.13–3.30 (m,6H), 3.36–3.52 (m, 8H), 3.55–3.64 (m,4H), 3.82(s,4H), 4.25 (s, 2H), 7.85 (m, 2H), 8.40 (t, J = 9.0 Hz, 1H), 8.60 (d, J = 6.0 Hz, 1H). ¹³C NMR (D₂O, 75 MHz) δ 49.9 (t, 2C), 50.8 (t), 51.2 (t), 54.6 (t), 55.4 (t), 55.5 (t), 57.0 (t), 126.26 (d), 126.8 (d), 141.3 (d), 147.1 (d), 152.4 (s), 172.7 (s), 174.6 (s). HRMS (Negative ion ESI) Calcd for C₂₀H₃₁N₅O₆ [M − H]⁻ m/z 436.2202. Found: [M − H]⁻ m/z 436.2232.

Computational studies.

All calculations were performed using Spartan’18 Parallel Suite.²⁰ A structure of Y(III)-NE3TA-PY or Lu(III)-NE3TA-PY complex was initially built and energy-minimized using the molecular mechanics force field (MMFF) model. MMFF conformational search for the energy-minimized Y(III)-NE3TA-PY or Lu(III)-NE3TA-PY complex were performed. Geometry optimizations of the major conformations for Y(III)-NE3TA-PY and Lu(III)-NE3TA-PY were performed in gas phase using density functional B3LYP (Becke, 3-parameter, Lee–Yang–Parr) model.^21–23 Atoms in the chelator (H, C, N, O) were described by using standard 6–31G* basis set,^24–25 while Y(III) and Lu(III) were modeled using the LANL2DZ (Los Alamos National Laboratory 2 double Z)-effective core potential (ECP) basis set.²⁶ The use of the LANL2DZ-ECP is based on the Dolg, Stoll, and Preuss ECP, where 4f¹⁴ electrons in the lanthanide are placed in the core.²⁷ The structure of the DFT-optimized Y(III)-NE3TA-PY or Lu(III)-NE3TA-PY conformer with energy minimum was calculated and visualized using the UCSF (University of California, San Francisco) molecular analysis program (Chimera 1.14)²⁸ or CYLview (Supporting information).²⁹ Molecular surface of Y(III)-NE3TA-PY and Lu(III)NE3TA-PY complexes was calculated using molecular surface method (MSMS) included in the UCSF molecular analysis program (Chimera 1.14).

Radiolabeling of NE3TA-PY with ¹⁷⁷Lu or ⁹⁰Y.

All HCl solutions were prepared from the commercially available ultra-pure HCl solution (JT baker, #6900–05). For metal-free radiolabeling, plasticware including pipette tips, tubes, and caps was soaked in 0.1M HCl overnight and washed thoroughly with Milli-Q (18.2MΩ) water and air-dried overnight. Ultra-pure ammonium acetate (Aldrich, #372331) was purchased from Aldrich and used to prepare 0.25M NH₄OAc buffer solutions (pH 5.5 or pH 7.0). After adjusting pH using 0.1M/1M HCl or NaOH solution, 0.25M NH₄OAc buffer solutions were treated with Chelex-100 resin (Biorad, #142–2842, 1g/100ml buffer solution), shaken overnight at room temperature, and filtered through 0.22μM filter (Corning, #430320) prior to use. ⁹⁰YCl₃ and ¹⁷⁷LuCl₃ were purchased from Perkin Elmer. Thin layer chromatography (TLC) plates (6.6 × 2 cm, Silica gel 60 F₂₅₄, EMD Chemicals Inc., #5554–7) with the origin line drawn at 0.6 cm from the bottom were prepared. To a buffer solution (0.25M NH₄OAc, pH 5.5 or pH 7.0) in a capped microcentrifuge tube (1.5 mL) was sequentially added a solution of NE3TA-PY (10 μg/ 1.6 μL H₂O). A solution of ⁹⁰YCl₃ or ¹⁷⁷LuCl₃ (0.05M HCl, 1.11 MBq, 30 μCi) was added to the aqueous solution of the chelator, and the total volume of the resulting solution was brought up to 20 μL by adding the buffer solution.¹² The reaction mixture was agitated on the thermomixer (Eppendorf, #022670549) set at 1,000 rpm at room temperature for 1 h. The labeling efficiency was determined by TLC eluted with a binary mobile phase (20 mM EDTA/ 0.15 M NH₄OAc). A solution of radiolabeled complexes (2 μL) was withdrawn at the designated time points, spotted on a TLC plate, and then eluted with the mobile phase. After completion of elution, the TLC plate was warmed and dried on the surface of a heater maintained at 35 °C and scanned using TLC scanner (Bioscan, #FC-1000). ⁹⁰Y-NE3TA-PY or ¹⁷⁷Lu-NE3TA-PY was detected at ~40 mm from the bottom of the TLC plate, while radionuclide ⁹⁰Y or ¹⁷⁷Lu bound to EDTA moved faster (~60 mm).

In vitro stability of ¹⁷⁷Lu-NE3TA-PY and ⁹⁰Y-NE3TA-PY.

Human serum was purchased from Gemini Bioproducts (#100110). ⁹⁰Y-NE3TA-PY and ¹⁷⁷Lu-NE3TA-PY was prepared by reaction of NE3TA-PY (50 μg/ 8.0 μL H₂O) with ⁹⁰Y (5.55 MBq, 150 μCi, 18.3 μL) or ¹⁷⁷Lu (5.55 MBq, 150 μCi, 3.46 μL) in 0.25M NH₄OAc buffer (pH 5.5, 88.54 μL for Lu-177 or pH 7.0, 73.7 μL for Y-90). Completion of radiolabeling was determined by TLC, and the resulting complexes ⁹⁰Y-NE3TA-PY or ¹⁷⁷Lu-NE3TA-PY were directly used for serum stability studies without further purification. ¹⁷⁷Lu-NE3TA-PY (5.48 MBq, 148 μCi, 99 μL) was added to human serum (500 μL) in a microcentrifuge tube. ⁹⁰Y-NE3TA-PY (5.03 MBq, 136 μCi, 99 μL) was added to human serum (500 μL) in a microcentrifuge tube. The stability of the radiolabeled complexes in human serum was evaluated at 37 °C for 7 days. The serum stability of the radiolabeled complexes was assessed by measuring the transfer of the radionuclide from each complex to serum proteins using TLC (eluent: 20 mM EDTA/ 0.15 M NH₄OAc). A solution of the radiolabeled complex in serum (5–16 μL for TLC) was withdrawn at the designated time point and evaluated by TLC. At each of the time points, the percentage of ¹⁷⁷Lu or ⁹⁰Y released from each of the radiolabeled complexes into serum was assessed by TLC. The radiolabeled complex ⁹⁰Y-NE3TA-PY or ¹⁷⁷Lu-NE3TA-PY was detected at ~40 mm from the bottom of the TLC plate, while unbound radionuclide ⁹⁰Y or ¹⁷⁷Lu moved faster (~60 mm).

EDTA (ethylenediaminetetraacetic acid) challenge of ¹⁷⁷Lu-NE3TA-PY and ⁹⁰Y-NE3TA-PY.

¹⁷⁷Lu-NE3TA-PY or ⁹⁰Y-NE3TA-PY was prepared by reaction of NE3TA-PY (10 μg/1.6 μL H₂O) with ¹⁷⁷Lu (1.11 MBq, 30 μCi, 0.68 μL) in 0.25M NH₄OAc buffer (17.72 μL, pH 5.5) or ⁹⁰Y (1.11 MBq, 30 μCi, 3.66 μL) in 0.25M NH₄OAc buffer (14.74 μL, pH 7.0). Completion of radiolabeling was determined by TLC, and the resulting complexes ⁹⁰Y-NE3TA-PY or ¹⁷⁷Lu-NE3TA-PY were directly used for EDTA challenge studies without further purification. A solution of EDTA (18 μL, 80 mM/H₂O, pH 7.0) at a 100-fold molar excess was added to a solution of ¹⁷⁷Lu-NE3TA-PY (0.99 MBq, 27 μCi/ 18 μL 0.25 M NH₄OAc buffer). A solution of EDTA (19 μL, 80 mM/H₂O, pH 7.0) at a 100-fold molar excess was added to a solution of ⁹⁰Y-NE3TA-PY (0.78 MBq, 21 μCi/ 19 μL 0.25 M NH₄OAc buffer). The resulting mixture was incubated for 24 hours at 37 ℃. The stability of ⁹⁰Y-NE3TA-PY or ¹⁷⁷Lu-NE3TA-PY complex in the presence of EDTA at a 100-fold molar excess was determined using TLC (eluent: 20 mM EDTA/0.15 M NH₄OAc).¹⁴ The radiolabeled complex ⁹⁰Y-NE3TA-PY or ¹⁷⁷Lu-NE3TA-PY was detected at ~40 mm from the bottom of the TLC plate, while unbound radionuclide ⁹⁰Y or ¹⁷⁷Lu moved faster (~60 mm).

Results and Discussion

Synthesis.

Synthesis of the new chelator NE3TA-PY is centered on reaction of N-tert-Butyl protected NE3TA (4) with 2-pyridylmethyl chloride (Scheme 1). Compound 3 was prepared from reaction of tert-Butyl protected NODA (1,4,7-triazacyclononane-1,4-diacetic acid) 1 using a modified synthetic method as we previously reported.¹⁸ Removal of the N-Benzyl group in compound 3 by hydrogenolysis was achieved to provide compound 4 in a nearly quantitative yield. Substitution reaction of compound 4 with 2-pyridylmethyl chloride produced compound 5 that was further treated with 6M HCl(aq) solution for acid-promoted deprotection of tert-Butyl groups to afford the desired chelator NE3TA-PY (6).

Scheme 1.

Synthesis of Chelator NE3TA-PY (6)

DFT calculations.

We conducted theoretical complexation studies of the new chelator NE3TA-PY with Lu(III) and Y(III) using density functional model. Geometries and relative energies of Lu(III)-NE3TA-PY and Y(III)-NE3TA-PY complexes in gas phase were calculated using the hybrid density functional B3LYP and Pople 6–31G* basis set. The most stable conformers with energy minimum for Y(III)-NE3TA-PY and Lu(III)-NE3TA-PY (Figure 2 and Supporting information) were obtained from the DFT calculations.

The chelator NE3TA-PY contains four tertiary nitrogens that are connected via ethylene chains and can form diastereomeric complexes with a metal cation. The four nitrogens (N1, N2, N3, and N4, Figure 2) in the chelator bound to Lu(III) or Y(III) to form four 5-membered chelate rings in two possible conformations (λ or δ). Each of the two pendant acetate attached to the TACN ring (N1 and N2) coordinated with the metal cation can adopt two different orientations (Δ or Δ helicity). Complexation of the tertiary nitrogen (N4) in NE3TA-PY with the metal cation leads to formation of non-interconvertible enantiomeric isomers (R or S configuration).

Figure 2. DFT-optimized structures for Lu(III)-NE3TA-PY and Y(III)-NE3TA-PY with energy minimum in gas phase.

All hydrogens are omitted for clarity, and carbon (grey), oxygen (red), nitrogen (blue), yttrium (grey), and lutetium (green) are highlighted in different color.

The DFT-optimized structures for both Lu(III)-NE3TA-PY and Y(III)-NE3TA-PY (Figure 2) were calculated to adopt the nitrogen atoms (N1-N3) of the TACN ring in a syn conformation and the pendant donor groups in Δ helicity and the four five-membered rings in the same gauche orientations (λλλλ). For the DFT-optimized Lu(III)-NE3TA-PY and Y(III)-NE3TA-PY complexes, the chiral nitrogen (N4) donor in the pendant arm is predicted to have R configuration. The DFT calculations on the relative free energies of the enantiomeric Lu(III)NE3TA-PY and Y(III)-NE3TA-PY complexes predicts that Δ(λλλλ)-N4(R) is slightly more stable than its enantiomeric counterpart Δ(δδδδ)-N4(S) (Supporting information). The oxygen atoms in the DFT-optimized Lu(III)-NE3TA-PY and Y(III)-NE3TA-PY complexes are more tightly coordinated to Lu(III) or Y(III) than the nitrogen atoms (mean distance of M-O and M-N, ~2.2Å vs 2.6Å, Table 1). The pyridyl ring in the chelator NE3TA-PY is predicted to adopt a favorable conformation for coordination to Lu(III) or Y(III), and the nitrogen atom in the pyridyl ring (N5) was estimated to make a tighter bond (distance of M-N5 bond, 2.475Å for Lu³⁺ and 2.545Å for Y³⁺) with Lu(III) or Y(III) than the nitrogen atoms in the tertiary amines (N1-N4, mean distance of M-N bond, 2.571Å for Lu³⁺ and 2.618Å for Y³⁺). Calculations on relative binding energies of the complexes predicts that Lu(III)-NE3TA-PY is more stable than Y(III)-NE3TA-PY by 8.797 kcal/mol (Table 1 and Supporting Information).

Table 1.

Distance (Å) of selected bonds in DFT-calculated structure of Lu(III)-NE3TA-PY and Y(III)-NE3TA-PY and their relative binding energy (kcal/mol).

Lu(III)-NE3TA-PY		Y(III)-NE3TA-PY
Lu-N1	2.610	Y-N1	2.596
Lu-N2	2.586	Y-N2	2.644
Lu-N3	2.525	Y-N3	2.614
Lu-N4	2.563	Y-N4	2.619
Lu-N5	2.475	Y-N5	2.545
Lu-N (Mean)	2.552	Y-N (Mean)	2.604
Lu-O6	2.193	Y-O6	2.265
Lu-O7	2.145	Y-O7	2.214
Lu-O8	2.172	Y-O8	2.250
Lu-O (Mean)	2.170	Lu-O (Mean)	2.243
Relative Binding Energy (kcal/mol)	0.000	Relative Binding Energy (kcal/mol)	8.797

Radiolabeling kinetics and in vitro serum stability.

The chelator NE3TA-PY was evaluated for radiolabeling efficiency with ⁹⁰Y and ¹⁷⁷Lu (Table 2). The new chelator in 0.25M NH₄OAc buffer solution (pH 5.5 or pH 7.0) was radiolabeled with ⁹⁰Y or ¹⁷⁷Lu at room temperature (RT). During the reaction time (1 h), the radiolabeling kinetics was determined by TLC analysis (Table 1 and Supporting Information). The data shown in Table 2 indicate that radiolabeling of NE3TA-PY with ¹⁷⁷Lu or ⁹⁰Y was significantly more efficient at pH 7.0 compared to pH 5.5. The rapid sequestration of the chelator with the radionuclides under neutral conditions may be explained by enhanced ligating capability of the basic nitrogen atom in the pyridyl ring. NE3TA-PY instantly and nearly completely bound to ¹⁷⁷Lu at the beginning of the reaction under neutral conditions (~93% labeling efficiency at 1 min, pH 7.0). NE3TA-PY was relatively slower in binding ⁹⁰Y (~59% at 1 min, pH 7.0) but almost completely sequestered ⁹⁰Y at 1 h time point (96%, pH 7.0, RT). ¹⁷⁷Lu-NE3TA-PY and ⁹⁰Y-NE3TA-PY were evaluated for complex stability in human serum. The radiolabeled complexes were freshly prepared and incubated in human serum (37 °C, pH 7) and determined for release of radioactivity to serum using TLC analysis (Figure 3 and Supporting Information). The data in Figure 3 show that ¹⁷⁷Lu-NE3TA-PY was stable in human serum for at least 7 days without a measurable loss of ¹⁷⁷Lu, while ⁹⁰Y-NE3TA-PY released a considerable amount of ⁹⁰Y (>12%) to serum over 7 days.

Table 2.

Radiolabeling efficiency of NE3TA-PY with ¹⁷⁷Lu or ⁹⁰Y (pH 5.5 and 7.0, RT)^*

Time	Radiolabeling efficiency (%)
	177Lu-NE3TA-PY		90Y-NE3TA-PY
	pH 5.5	pH 7.0	pH 5.5	pH 7.0
1 min	38.4 ± 2.4	93.0 ± 1.3	11.3 ± 0.5	58.8 ± 1.8
10 min	94.8 ± 0.4	97.7 ± 0.6	53.4 ± 0.2	87.1 ± 0.6
30 min	99.5 ± 0.4	99.0 ± 0.4	82.1 ± 0.1	93.2 ± 0.1
60 min	99.6 ± 0.3	99.4 ± 0.0	92.6 ± 0.8	96.1 ± 0.1

^*Radiolabeling efficiency (mean ± standard deviation%) was measured in duplicate using TLC eluted with a binary mobile phase (20 mM EDTA/0.15 M NH₄OAc).

Figure 3.

In vitro complex stability of ¹⁷⁷Lu-NE3TA-PY and ⁹⁰Y-NE3TA-PY in human serum at pH 7 and 37 °C.

The radiolabeled complexes ¹⁷⁷Lu-NE3TA-PY and ⁹⁰Y-NE3TA-PY were further assessed for complex stability in the presence of EDTA. ¹⁷⁷Lu-NE3TA-PY or ⁹⁰Y-NE3TA-PY was incubated with EDTA at a 100-fold molar excess and monitored for release of radioactivity using TLC analysis (Figure 4 and Supporting Information). When challenged by EDTA, ¹⁷⁷Lu-NE3TA-PY remained stable and lost a minimum amount of ¹⁷⁷Lu in 48 h (~2.5%). However, ⁹⁰Y-NE3TA-PY was shown to undergo transchelation with EDTA and released a significant amount of ⁹⁰Y bound to EDTA (~30% at 24 h post-incubation time).

Figure 4.

Complex stability of ¹⁷⁷Lu-NE3TA-PY and ⁹⁰Y-NE3TA-PY in EDTA solution (100-fold molar excess) at pH 7 and 37 °C.

The radiolabeling and in vitro complex stability data indicate that NE3TA-PY with potential 8 donor groups displayed excellent radiolabeling kinetics and stability with ¹⁷⁷Lu. The chelator was slower in binding to ⁹⁰Y under mild radiolabeling conditions to form a less stable complex with ⁹⁰Y. While both ¹⁷⁷Lu and ⁹⁰Y have the prevalent oxidation state (+3), ¹⁷⁷Lu has a smaller ionic radius than ⁹⁰Y (0.977Å vs 1.019Å for coordination number 8).³⁰ As predicted by DFT calculations on Lu(III)-NE3TA-PY and Y(III)-NE3TA-PY complexes, the small macrocyclic cavity (TACN) is more tightly coordinated with Lu(III) than Y(III) (Table 1). Lu(III) is more closely bound to the aromatic amine in the pyridyl ring (Lu-N5 distance: 2.475Å vs 2.545Å) than Y(III). The pyridyl ring in Y(III)-NE3TA-PY complex is postulated to cause a considerable ligand strain on Y(III)-coordination sphere, leading to the formation of less stable Y(III)-NE3TA-PY complex.

Conclusion

The novel chelator NE3TA-PY containing three aminocarboxylates and a pyridyl ring as donor groups was synthesized and evaluated for complexation with ¹⁷⁷Lu and ⁹⁰Y. The radiolabeling and in vitro complex stability data confirm that the new chelator produced excellent radiolabeling kinetics and complex stability profiles with ¹⁷⁷Lu. The chelator NE3TA-PY was relatively slower in binding ⁹⁰Y, and the corresponding ⁹⁰Y-NE3TA-PY complex was relatively less stable than ¹⁷⁷Lu-NE3TA-PY in human serum and EDTA solution. The results of the DFT calculations also suggest that the chelator NE3TA-PY forms more stable complex with Lu(III) than Y(III). The experimental and computational data suggest that NE3TA-PY is a promising octadentate ligand that can be further structurally modified and studied for conjugation to a bioactive molecule for targeted radionuclide therapy and imaging.

Highlights.

• NE3TA-PY ((7-[2-[(carboxymethyl)(2-pyridylmethyl)amino]ethyl]-1,4,7-triazacyclononane-1,4diacetic acid).

• Synthesis of a chelating agent (NE3TA-PY) for formation of a neutral metal complex.

• Comparative DFT calculations of Lu(III)-NE3TA-PY and Y(III)-NE3TA-PY.

• Rapid formation of stable ₁₇₇Lu-NE3TA-PY complex under mild radiolabeling conditions.

• Excellent complex stability of ₁₇₇Lu-NE3TA-PY in human serum and EDTA solution.